Alternatives to antibiotics in poultry feed: molecular ... · livestock; however, a 33% increase in the therapeutic use of antibiotics by farmers was reported subsequently (Ferber

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Critical Reviews in Microbiology

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Alternatives to antibiotics in poultry feed:molecular perspectives

Gayatri Suresh, Ratul Kumar Das, Satinder Kaur Brar, Tarek Rouissi, AntonioAvalos Ramirez, Younes Chorfi & Stephane Godbout

To cite this article: Gayatri Suresh, Ratul Kumar Das, Satinder Kaur Brar, Tarek Rouissi,Antonio Avalos Ramirez, Younes Chorfi & Stephane Godbout (2018) Alternatives to antibioticsin poultry feed: molecular perspectives, Critical Reviews in Microbiology, 44:3, 318-335, DOI:10.1080/1040841X.2017.1373062

To link to this article: https://doi.org/10.1080/1040841X.2017.1373062

Published online: 11 Sep 2017.

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Alternatives to antibiotics in poultry feed: molecular perspectives

Gayatri Suresha, Ratul Kumar Dasa,b�, Satinder Kaur Brara, Tarek Rouissia, Antonio Avalos Ramirezc,Younes Chorfid and Stephane Godboute

aINRS-ETE, Universit�e du Qu�ebec, Qu�ebec, QC, Canada; bTERI Deakin Nanobiotechnology Centre, TERI Gram, The Energy and ResourcesInstitute, Gurgaon, India; cCentre National en �Electrochimie et en Technologie Environnementales Inc, Shawinigan, Canada;dD�epartement de biom�edecine v�et�erinaire, Universit�e de Montr�eal, St-Hyacinthe, Canada; eInstitut de recherche et de d�eveloppementen agroenvironnement, Qu�ebec, Canada

ABSTRACTThe discovery of the growth promoting property of antibiotics led to their use as antibiotic feedadditives (AFAs) in animal feed at sub-therapeutic doses. Although this has been beneficial foranimal health and productivity, it has been, essentially, a double-edged sword. The continuedand non-judicious use of AFAs has led to the selection and dissemination of antibiotic-resistantstrains of poultry pathogens such as Salmonella, Campylobacter and Escherichia coli. The rapidspread of drug-resistant pathogens as well as emergence of antibiotic-related environmental pol-lutants is of global concern. Hence, the identification and development of new and effectivealternatives to antibiotics that do not hinder productivity is imperative. For this, it is essential tounderstand not only the molecular basis of development of resistance to AFAs but also themechanisms of action of AFA alternatives and how they differ from AFAs. This review provides amolecular perspective on the alternatives to antibiotics that have been proposed till date andtheir current trends, as well as novel approaches such as development of improved deliverysystems.

ARTICLE HISTORYReceived 20 February 2017Revised 17 July 2017Accepted 25 August 2017Published online 11 Septem-ber 2017

KEYWORDSMultidrug resistance;alternatives to antibioticfeed additives; molecularmechanisms; antibacterial;immunomodulation

Introduction

For almost eight decades, antibiotic feed additives(AFAs) have been used in poultry for increased prod-uctivity and efficiency. It has been estimated that by2030, a total of 105,596 (±3605) tons of antimicro-bials will be consumed in food animal productionglobally (Van Boeckel et al. 2015). Several studiesindicate that the use of antimicrobials has resulted inincreased productivity and decreased cost for con-sumers (Ricke et al. 2012). The primary function ofAFAs in poultry is as growth promoters. The adminis-tration of AFAs at sub-therapeutic dosages has beenshown to increase growth rate, feed conversion andconsequently, broiler performance. Additionally, AFAsare also used for the treatment of infections causedby common poultry pathogens such as Salmonella,Campylobacter, Escherichia coli, Listeria and Clostridium(Jorgensen et al. 2002; Bohaychuk et al. 2006; Hafez2011). The antimicrobial activity of these drugs is dueto the different targets they attack, such as cell wall,cell membrane, protein synthesis and DNA replication.

Some of the antimicrobials used as feed additivesin poultry are penicillin, neomycin, erythromycin,chlortetracycline, oxytetracycline, streptomycin, chlor-amphenicol and fluoroquinolones (McEwen andFedorka-Cray 2002).

Despite their substantial contribution to the poultryindustry, AFAs are under surveillance due to an increasein the incidence of drug resistance, caused majorly bythe use of these drugs by livestock farmers without vet-erinary consultation or proper directions for dosage(Bbosa and Mwebaza 2013). The prolonged and unregu-lated use of these antimicrobials has led to the selectionof drug resistant strains of pathogens. For example, aprimary cause of salmonellosis in humans – an MDR(multidrug resistant) strain of Salmonella typhimuriumwas first identified in livestock (Woc-Colburn and Bobak2009). According to studies done by Iovine and Blaser(2004) and Tambur et al. (2010), emergence of drugresistant strains of C. jejuni causing disease in humanscan be associated with the use of antimicrobials inpoultry farms. Cross resistance to antibiotics used in

CONTACT Satinder Kaur Brar [email protected] INRS-ETE, Universit�e du Qu�ebec, 490 Rue de la Couronne, Qu�ebec, QC, Canada G1K 9A9�Present address: TERI Deakin Nanobiotechnology Centre, TERI Gram, The Energy and Resources Institute, Gual Pahari, Faridabad Road, Gurgaon, Haryana122 001, India� 2017 Informa UK Limited, trading as Taylor & Francis Group

CRITICAL REVIEWS IN MICROBIOLOGY, 2018VOL. 44, NO. 3, 318–335https://doi.org/10.1080/1040841X.2017.1373062

humans can also develop due to their chemical similar-ities with veterinary antibiotics.

The Swann committee report released in UnitedKingdom in 1960 raised serious concern worldwide overthe safety of the use of AFAs in animal industry. Theoxy tetracycline resistance of humans was found to betransferred from animal meat and this had led to restric-tion or ban in uses of AFAs. As more cases of bacterialantibiotic resistance (through transfer from animal tohuman) were reported from different parts of the world,the controversy over the use of AFAs became a publicconcern by the end of 1990. Due to the long term rami-fications of AFAs, they were banned in Sweden in 1986.From 1997 to 2006, the European Union (EU) phasedout the use of AFAs in animal feed and veterinary pre-scriptions (Cogliani et al. 2011). The Netherlands gov-ernment set a target of 70% reduction in the use ofAFAs by 2015. Food companies such as McDonalds andTyson Foods have announced their future policies onsourcing of chickens from antibiotic free poultry farms.The restriction or ban on the use of AFAs in Europe hasprimarily resulted in the reduction in actual exposure toantibiotics. Scientifically, this trend has been evaluatedby calculating the Animal Level of Exposure toAntimicrobials index (ALEAI). The estimation of ALEAI isdone from different factors such as PopulationCorrection Unit and dose (concentration x time) of anti-biotics. At the first Poultry Summit Europe (17–18 May2016, Utrecht, The Netherlands), the participants sug-gested different strategies such as biosecurity, data inbreeding and hygiene in the feed mill for antibioticreduction in poultry industry (Koeleman 2016).

However, this ban has been at the cost of productiv-ity, animal welfare and subsequently, an increase in theveterinary use of therapeutic antibiotics has been docu-mented (Casewell et al. 2003). This was seen inDenmark, where, following the ban of AFAs, there wasalmost a 30% reduction in drug resistant strains fromlivestock; however, a 33% increase in the therapeuticuse of antibiotics by farmers was reported subsequently(Ferber 2003). In North America, antibiotics continue tobe used as feed additives. However, in 2005, enrofloxa-cin and sarfloxacin – both fluoroquinolones – werebanned from use in poultry in the United States,followed by a ban on the extra-label usage of ceftiofur(a cephalosporin) (Prescott and Dowling 2013). Unlikethe EU and the United States, Canada has no formalrestraints on the use of AFAs, and veterinarians inCanada can prescribe antibiotics for extra-label use(Maron et al. 2013; Prescott and Dowling 2013). Withthe global consumption of poultry increasing at anannual rate of more than 3.6% (Revell 2015), it has

become imperative to develop feasible alternatives tofeed antibiotics without compromising on productivity.

Drug resistance versus efficacy of antibioticfeed additives: molecular domain

Resistance to antimicrobials can be facilitated throughmechanisms such as reducing outer membrane perme-ability by downregulation of porins to decreased druguptake, genetic modification of target by mutation,methylation of target to prevent antibiotic binding,enzymatic inactivation of drug, bypassing pathwaysaffected by the drug or overexpression of efflux pumpsfor active transport of drugs out of cell (Atterbury et al.2011). The field of molecular basis of antibiotic resist-ance has been garnering interest. In recent times, sev-eral genes have been found to be associated with thedevelopment of drug resistance. Bacterial resistance toAFAs can be intrinsic or acquired. Intrinsic resistanceoccurs due to random mutations in the bacterialchromosome, and is transmitted vertically to progenycells. Extrinsic resistance can be attributed to bacterialmechanisms of horizontal gene transfer (HGT) whichcan transfer resistance genes to other bacteria (e.g.known human pathogens) (Diarra and Malouin 2014;Toutain et al. 2016). The methyl-directed mismatchrepair (MMR) pathway (coded for by the “Mut” genes) isa post-replication DNA repair system that specificallytargets mismatched bases, thus ensuring fidelity of DNAreplication, and preventing recombination between dis-similar bacterial species. Therefore, bacteria with aninactivated MMR system are prone to both spontaneousmutation and recombination with diverged species(LeClerc et al. 1996). The spread of antibiotic resistancegenes can majorly be attributed to HGT. There are threemechanisms for HGT: (i) transformation, in which extra-cellular DNA is taken up by competent cells; (ii) trans-duction, in which gene transfer is facilitated throughbacteriophages; and (iii) conjugation, in which DNA istransferred via a physical contact between a donor andrecipient cell (von Wintersdorff et al. 2016). The plas-mid-mediated dissemination of drug resistance genesvia conjugation is the major cause of the current magni-tude of dissemination of drug resistance. Plasmids areautonomously replicating extrachromosomal DNA mol-ecules which either inherently carry genes coding fordrug resistance or mobilize these genes from bacterialchromosomes by recombination. Additionally, cluster-ing of several resistance genes on a single plasmidcan lead to selection of multidrug resistant strains via asingle “horizontal transfer event” (Barlow 2009).Conjugative plasmids have genes which code for forma-tion of a pilus between the donor and the

CRITICAL REVIEWS IN MICROBIOLOGY 319

lipopolysaccharide and outer membrane proteins of therecipient cell. A junction is formed between the twocells for transport of the DNA and a pore formed in therecipient cell facilitates the entry of the DNA into it(Thomas and Nielsen 2005). Transposons are alsomobile genetic elements which can be transferred viaconjugation and are implicated in spread of antimicro-bial resistance (von Wintersdorff et al. 2016). Thus, HGTmechanisms play a very important role in creating newresistant strains of pathogens.

Additionally, antibiotic-related contaminants (anti-microbial resistant pathogens, antimicrobial resistancegenes and antibiotic drugs) are classified as emergingenvironmental pollutants (Keen and Patrick 2013). Soiland water resources are the main reservoirs of antibioticresistance. Antibiotics added in animal feed or drinkingwater are not completely metabolized in the gut ofpoultry and about 70% is the drug is excreted in itsunmetabolized form (K€ummerer 2009). Animal wastescontaining residual AFAs, when used as manure, canintroduce antibiotics, resistant bacteria and antibioticresistance genes in the soil ecosystem (Furtula et al.2010; Franklin et al. 2016). Recently, Ho et al. (2014)reported the high concentration of flumequine(1.3mg/kg dry weight) in soil samples that were treatedwith broiler contaminated with flumequine, doxycyclineand trimethoprim among others. Antibiotics in soil caneither get degraded within a few hours or days, or maypersist in soil for several months to years (Jechalke et al.2014), depending on soil parameters, temperature, pHand adsorption of antibiotics to organic matter inmanure (Tasho and Cho 2016). Therefore, an excessiveuse of AFAs can lead to their accumulation in the soiland cause significant alteration of soil microbiota.Qingxiang et al. (2009) showed that exposure to oxy-tetracycline had an adverse effect on the structure andactivity of the rhizosphere soil microbial community.Studies have also documented the accumulation ofantibiotics in plants for which poultry litter was used asmanure (Kumar et al. 2005; Tasho and Cho 2016).Residual AFAs in soil promote selective proliferation ofresistant strains and increase the diversity of drug resist-ance genes which can be subsequently be transferredvertically or horizontally (Ding and He 2010; Jechalkeet al. 2014). These pollutants can also enter ground andsurface water resources from contaminated soils byleaching or water run-off through livestock wastewater,and affect the microbial community composition (BarraCaracciolo et al. 2015). In aquatic environments, antibi-otics may get degraded (fully or partially), or may accu-mulate in tissues of aquatic organisms (Gaw et al. 2014),that could subsequently be consumed by humans. Thedissemination of antibiotic resistance in the

environment (from “farm-to-fork”) has been depicted inFigure 1.

The mechanisms of the growth promotion by AFAsare not completely understood and are thought to beby inhibition of subclinical infections, reduction ofmetabolites which can affect growth such as end prod-ucts of bile degradation, reduction of nutrient availabil-ity to pathogens, thinning of intestinal epithelium andexhibition of anti-inflammatory action on macrophagesand granulocytes (Br€ussow 2015). The poultry gut is acomplex and dynamic microbiome, with the ratio ofmicrobes to host cells being approximately 10:1.Gaskins et al. (2002) proposed that the growth promo-tion by AFAs is primarily due to inhibition of the normalgut microbiota which would increase the host nutrientavailability as well as reduce maintenance costs, i.e. a“bacteria-centric” effect. Studies have also shown thatdecrease in the gut microbiota improves fat utilizationas these bacteria can degrade bile salts leading todecreased digestibility of fat. Gut microbiota are alsoknown to increase thickness of the gut leading toreduced nutrient absorption, as well as, elicit hostimmune response due to their antigenic determinantswhich is an energy utilizing process (Milanov et al.2016). Another school of thought states that growthpromotion by the sub therapeutic use of AFAs due toantibiotic activity is unlikely, and rather, can be attrib-uted to their “host-centric” effect, i.e. immunomodula-tory effect in the host. Niewold et al. stated that theeffect of AFAs is “growth-permitting” rather than“growth promoting”, and is due to their ability to inhibitintestinal inflammation (thus preventing thickening ofthe intestine wall, as well as waste of energy ratherthan an antimicrobial mechanism (Niewold 2007).Hence, an ideal alternative to AFAs would interact withimmune cells that modulate inflammatory response,and/or would target a site different from that of con-ventionally used antibiotics for antimicrobial activity,thus evading the selection of drug resistant strains.Several alternatives to AFAs, having similar effects inanimals, have been proposed (Table 1), and their effectsin broiler performance have been evaluated (Table 2).

Most of the proposed AFA alternatives primarily havea “host centric” mechanism of action, which makes itless likely for the development of resistance againstthem. It is essential to study the molecular basis of themechanism of the growth promotion activity of theAFAs as well as the potential alternatives (Table 3) forthe identification of suitable alternatives that differ fromthe default modalities either in their target or theirmechanism of action to evade the development ofresistance, but would have effects similar to the antibi-otics in the host, whether it is reduction of pathogen

320 G. SURESH ET AL.

load or immunomodulation (Allen et al. 2013; Rios et al.2016). Although there is extensive literature on molecu-lar mechanisms of bacterial resistance and proposedmechanisms of AFAs, there is no comprehensive reviewof the molecular basis of the mode of action of thealternatives to AFAs. This review attempts to bridge thisgap in knowledge, for the development of an effectivealternative to feed antibiotics (Figure 2).

Phytogenic feed additives (PFAs)

Plant secondary metabolites or phytochemicals areorganic bioactive compounds produced by plants dur-ing their normal metabolism and may or may not havenutritional value (Hashemi and Davoodi 2010, 2011).They are known to have antioxidative and immunomo-dulatory properties, and are thought to increase nutri-ent digestion and absorption in the gut (Ahmed et al.2013). Their antimicrobial action is suggested to be dueto mechanisms such as disruption of pathogen mem-branes, affecting virulence by modification of cell

surface, activation of cells of the immune system, pro-motion of beneficial bacteria in the gut (Diaz-Sanchezet al. 2015). A study by Abudabos et al. (2016) reportedthat in broilers challenged with S. typhimurium, phyto-genic feed additives were found to have an effect simi-lar to avilamycin on the growth and blood biochemicalprofile. Plant secondary metabolites have also beenstudied for their immunomodulation effects, which arethought to be due to (i) induction of heat shock pro-teins which increase the efficiency of protein transla-tion, (ii) induction of Toll-like receptors (TLRs) whichrecognize conserved microbial molecules and activateimmune response in the host and (iii) inducing the pro-liferation of Th-1 and Th-2 cells (subtypes of T-Helpercells) to maintain the balance between cellular andhumoral branches of immunity (Hashemi and Davoodi2012). Phytogenic feed additives have also been shownto increase livestock productivity by increasing feed effi-cacy by the stimulation secretion of intestinal mucus,saliva and bile, as well as by inducing morphologicalchanges such as increase in villi and crypt size in the

Figure 1. Spread of antibiotic resistance in environment.

CRITICAL REVIEWS IN MICROBIOLOGY 321

avian intestine (Banerjee et al. 2013; Yitbarek 2015).Phytobiotics are a complex blend of organic molecules,and the multiple active components could each have adifferent mode of antimicrobial action. Due to this, itwould be more difficult for bacteria to develop resist-ance to them.

Essential oils (EO) have been studied extensively asan alternative to AFAs. EOs are volatile, aromatic, lipo-philic compounds, composed primarily of terpenes andphenylpropenes (Hengl et al. 2011). Antibacterial prop-erty of EOs could be attributed to their lipolytic prop-erty, and their hydrophobic components, thatcompromise the bacterial cell membrane structure byaltering its permeability to ions (Helander et al. 1998),leading to disruption of bacterial enzyme system,decrease in bacterial growth and “cell leakage”. A studyby Kollanoor et al. (2012) showed the efficiency ofeugenol and trans-cinnamaldehyde to reduceSalmonella enteritidis colonization in chicken ceca bydownregulation of the motility and virulence genes.

Apart from EOs, PFAs also include tannins, and sap-onins herbs, spices, oleoresins, flavonoids and alka-loids (Patra and Saxena 2009), with differentmechanisms of antimicrobial action. Tannins can causeiron deprivation or interact with specific cellularenzymes, saponins form complexes with membranesterols leading to membrane damage, and alkaloidsinhibit topoisomerase and interrupt DNA synthesis(Hashemi and Davoodi 2011)

Dietary inclusion of PFAs is also known to be benefi-cial due to their antioxidant property (which is thoughtto protect lipids in feed from oxidation), reduction ofoxidative stress, immune cell proliferation, increasedproduction of intestinal and pancreatic enzymes, andincreased cytokines production, and anti-inflammatoryproperties (Alloui et al. 2014; Gadde et al. 2017). A studyby Khadem et al. (2014) made use of extracts fromMacleaya cordata (which is rich in anti-inflammatoryalkaloids, such as sanguinarine and chelerythrine), as afeed additive and found that the plant extract downre-gulated the expression of inducible NO synthase, thusexplaining the anti-inflammatory effect.

Some components of EOs can stimulate the secretionof digestive enzymes like trypsin, amylase and lipase byinteracting with cellular receptors in the pancreas,which can increase feed intake and feed conversionratio (Valientes 2014). A blend of essential oils ofpeppermint, anise, clove and caraway has been eval-uated for its anti-inflammatory and antioxidative prop-erties. It was shown to inhibit factors responsible forinflammation (interleukin 8, monocyte chemotactic pro-tein and intracellular adhesion molecule) by the down-regulation of a transcription factor responsible forexpression of these genes, and also activate the cellularpathways which induced genes for defence againstreactive oxygen species (Steiner and Syed 2015). InSeptember 2015, a phytonutrient formulation, CCC (car-vacrolþ cinnamaldehydeþ capsicum oleoresin) was

Table 1. Comparison of Antibiotic Feed Additives and proposed alternatives to AFAs.Alternatives to AFAs

Antibioticfeed

additivesPhytogenic

feed additives Probiotics Prebiotics Feed acidifiers AMPs Bacteriophages Antibodies

Directantibacterialactivity

Yes Yes No No Yes Yes Yes Yes

Immuno-modulation

Yes Yes Yes Yes Yes Yes Yes Possible

Proliferation ofbeneficialbacteria

No Yes Yes Yes Yes No No No

Nutrientabsorption

Increased due tothinning ofepithelial wall

Increased due toinduction ofdigestive enzymes

Increased dueto inductionof digestiveenzymes

Unknown Increased dueto gutacidificationand increasedproteaseactivity

Thought toincrease

Unknown Unknown

Development ofresistance

Very common Could be difficultdue to multipletargets of action

No, due toindirectantimicrobialactivity

No, due toindirectantimicro-bialactivity

Not verycommon

Not very com-mon as it isnot energyefficient forcell

Yes, but morethan onephage avail-able for sin-gle host

No

Stability Stable Stable Stable Stable Stable Vulnerable toproteolysis

Could be vul-nerable tolow pH ingut

Vulnerable toheat andproteolysis

322 G. SURESH ET AL.

Table 2. Evaluation of effects of AFA alternatives in broiler performance.AFA alternative Effect in poultry References

Phytogenic feedadditives

(i) Increase in body weight(ii) Improvement in feed conversion ratio and carcass yield(iii) Decrease in pathogen counts(iv) Improvement of fatty acid profile in egg yolk(v) Increased serum proteins and antioxidant status

(Bernard et al. 2016; Peng et al. 2016)(Jahan et al. 2016; Sadeghi et al. 2016)(Chang et al. 2016; Lan et al. 2016)(Raza et al. 2016)(Alzawqari et al. 2016)

Probiotics (i) Increase in body weight and feed conversion(ii) Decrease in pathogen count, increase of beneficial bacteria in gut

(Gheisar et al. 2016; Hatab et al. 2016)(Olnood et al. 2015; Li et al. 2016)

Prebiotics (i) Increase in disease resistance, broiler efficiency and nutrient availability(ii) Increase in weight and population of beneficial bacteria(iii) Decrease in pathogen count(iv) Reversal of coccidial lesions

(Ganguly 2015)(Arsi et al. 2015; Pourabedin and Zhao 2015)(Kim et al. 2011; Shang et al. 2015)(Chand et al. 2016)

Feed acidifiers (i) Decrease in pathogen count(ii) Improvement in body weight gain ad feed conversion ratio(iii) Improvement of phytate phosphorus utilization(iv) Decrease in mortality and feed cost, increase in dressing percentage and liver weight

(Koyuncu et al. 2013; Sultan et al. 2015)(Sohail et al. 2015; Reda et al. 2016)(Rafacz-Livingston et al. 2005)(Khan et al. 2016)

AMPs (i) Decrease in pathogen counts(ii) Increase in beneficial bacteria, nutrient absorption, weight gain and feed conversion ratio

(Messaoudi et al. 2012; Wang et al. 2015)(Wang et al. 2011; Aguirre et al. 2015)

Bacteriophages (i) Prevention of diseases in birds(ii) Decrease in pathogen count(iii) Improvement in weight gain and feed conversion ratio

(Kim et al. 2013; El-Gohary et al. 2014)(Hungaro et al. 2013; Kittler et al. 2013)(Miller et al. 2010)

Antibodies (i) Lowering fecal shedding of and cecal colonization by pathogen(ii) Improving feed efficacy and intestinal health of birds

(Al-Adwani et al. 2013; Hermans et al. 2014)(Mahdavi et al. 2010)

Table 3. Mechanisms for growth promotion of proposed alternatives to AFAs.Feed additives Antibiotic activity Immunomodulatory activity

Phytogenic feed additives (i) Disruption of membranes and enzyme systems due tolipolytic activity

(ii) Modification of cell surface(iii) Inhibition of DNA synthesis(iv) Downregulation of virulence genes

(i) Induction of heat shock proteins to increase proteintranslocation

(ii) Activation of Toll-like receptors and Th cellproliferation

(iii) Stimulation of mucus, bile etc by intestinalepithelium

Probiotics (i) Competitive exclusion by decrease of pH, andcompeting for nutrients and attachment sites

(ii) Induce production of antimicrobial peptides byepithelium

(i) Proliferation of immune cells such as macrophages,monocytes, NK cells, T cells, etc.

(ii) Increase production of immunoglobulins, cytokines,and reactive oxygen and nitrogen species

(iii) Enhance mucin production by intestinal epitheliumto prevent bacterial translocation

Prebiotics (i) Production of antimicrobial compounds on fermenta-tion

(ii) Binding to bacterial surface receptors to preventadhesion to intestinal epithelium

(iii) Production of host antimicrobial peptides

(i) Activation of macrophages/dendritic cells by inter-action with specific receptors on cell surface

(ii) Increase mucin and goblet cell production by gutepithelium

(iii) Downregulation of proinflammatory cytokines,oxidative stress

Feed acidifiers (i) Inhibition of enzyme system and disruption ofmembrane structure and cell turgidity by decreasingcytoplasmic pH

(ii) Disruption of DNA, transcription and translation

(i) Elicit faster immune response by increase in CD4 andlymphocyte cell counts

(ii) Inhibition of proinflammatory pathways

AMPs (i) Altering membrane permeability, leading to cell lysisby transmembrane pore formation

(ii) Inhibition of cell cycle, activation of lytic enzymes,production of free radicals

(i) Proliferation of immune cells and production ofcytokines

(ii) Mast cells stimulation leading to vasodilation(iii) Induction of wound repair mechanisms

Bacteriophages (i) Lysis of bacterial cells by specific lytic phages(ii) Lysis of peptidoglycan-cell wall by phage encoded

enzymesAntibodies (i) Binding to cell surface receptors to prevent gut

adherence and colonization(ii) Agglutination, increased phagocytosis by surface

modification

CRITICAL REVIEWS IN MICROBIOLOGY 323

officially approved as the first 100% botanical zoo tech-nical additive to increase growth in broilers in Europe.Data gathered from 20 years of field trials conducted indifferent parts of the world confirmed the efficacy ofCCC as potential alternative to default modalities suchas avilamycin, bacitracin, flavophospholipol, or enramy-cin (Finding alternatives to antibiotics 2016). The immu-nomodulation effect of CCC was shown by Lillehoj et al.(2011), who reported the downregulation of expressionof oxidative stress, and the upregulation of inflamma-tory cytokines and genes associated with metabolic andendocrine systems in chicken.

Probiotics

The molecular mechanisms of the probiotic–hostinteraction of immunomodulation are not completelyunderstood. Proposed mechanisms include increase incell-mediated immune response, TLR signalling andantibody production and decrease of cellular apoptosis,etc. (Khan et al. 2016). Host intestinal epithelial cells anddendritic cells have certain receptors (e.g. TLRs, nucleo-tide-binding oligomerization domain (NOD) proteins)

which are activated by probiotic MAMPs (microorgan-ism-associated molecular patterns) such as fimbriae,flagella, lipopolysaccharide, lipoteichoic acid and pep-tidoglycan. Activation of these receptors leads to induc-tion of signal transduction pathways in the host cell fortranscription of genes coding for chemokines and cyto-kines, which can subsequently stimulate host systemicand mucosal immunity (Ajithdoss et al. 2012; Hardyet al. 2013). IL-12, a pro-inflammatory cytokine, andIL-10, an anti-inflammatory cytokine, are of particularinterest with respect to probiotics. Immunostimulatoryprobiotics induce IL-12 proliferation, which in turnincreases potency of Natural Killer (NK) cells and indu-ces T helper pathways. Immunoregulatory probioticsinduce IL-10 proliferation which then induces T regula-tory pathway (Yaqoob 2014).

Probiotics have also been known to alter the gut epi-thelial architecture. The mucus layer (composed of aclass of glycoproteins known as mucins) along with thegut epithelium forms the first line of host defence.Studies have shown that certain probiotic speciesincrease the expression of MUC-2 and MUC-3 geneswhich code for synthesis of mucins by goblet cells.

Figure 2. Overview of mechanisms of alternatives to antibiotic feed additives.

324 G. SURESH ET AL.

Increased mucus production in the gut prevents theadherence and subsequent colonization of the intestinalepithelium by pathogenic bacteria (Smirnov et al. 2005;Hardy et al. 2013). Additionally, probiotics can alsomaintain the integrity of epithelial tight junctions byupregulating genes that code for junction proteinswhich are responsible for tight junction signalling, aswell as restoration of mucosal integrity (Syngai et al.2016). Seth et al. (2008) reported that probiotic pro-duced proteins protect tight junctions from H2O2

induced disruption by the activation of Protein kinase Cisoforms and mitogen-activated protein (MAP) kinase.

Several mechanisms have been reported for toxininhibition by probiotic species, such as production ofa protease to degrade the toxin and its receptor,reduction of cyclic Adenosine MonoPhosphate (cAMP)production which eventually prevents diarrhoea andbinding of the toxin to receptors on the probioticsurface (Khan and Naz 2013).

Probiotics do not exert a direct antimicrobial effecton pathogenic bacteria in the gut, rather they employcompetitive exclusion (CE) to prevent pathogen colon-ization. This along with the complex, multiple mecha-nisms of action of probiotics makes it difficult todevelop bacterial resistance to probiotics.

Prebiotics

The proposed mechanisms of action for prebioticsinclude blocking receptor sites for pathogen adhesion,immunomodulation, production of antimicrobial com-pounds on fermentation and modifying gut morph-ology (Pourabedin and Zhao 2015). Immunomodulationby prebiotic is thought to be due to activation of innateimmunity by the interaction of the sugars with certainreceptors present on surface of dendritic cells and mac-rophages, which can then stimulate production of cyto-kines, proliferation of lymphocytes and activity of NKcells (Hashim 2012; Saad et al. 2013).

Extensive studies have been carried out on mannanoligosaccharide (MOS), obtained from the outer cellwall of S. cerevisiae. Mannans, present in MOS bind tomannose-specific type-I fimbriae on Gram-negativepathogens (such as E. coli and Clostridium), thus pre-venting them from adhering to, and colonizing the gut(Sinovec and Markovi�c 2005). Xiao et al. reported thealteration of expression of several genes – APOA1 andLUM – in the bird jejunum by dietary supplementationof MOS. They also reported the association of MOS withincreased expression of genes responsible for oxidativephosphorylation, mitochondrial electron transport chainand antioxidant enzymes (Xiao et al. 2012). MOS havealso been shown to affect goblet cell number and

mucin production in host gut, as well as host mRNAexpression, although the mechanism for this is not clear(Cheled-Shoval et al. 2014). Avian immunity can also beimproved by MOS by the activation of macrophagesthrough mannose-specific receptors (Hashim 2012).Shao et al. (2016) reported that supplementation ofb-glucans in broiler diets stimulated the synthesis ofhost antimicrobial peptides against Salmonella infec-tion. Dietary supplementation of inulin in broilers hasbeen shown to upregulate transcription of genes forimmune system, cholesterol synthesis and bile produc-tion in chicken (Tsurumaki et al. 2015). In another study,Babu et al. (2012) reported that inulin treatment candecrease the expression of IL-1b, a pro inflammatorycytokine, thus preventing IL-1b associated cell death ofmacrophages, and aiding in antibacterial effect. In amicroarray-based gene expression study, Sevane et al.observed a modification of the liver transcriptome pro-file of chicken supplementation of feed with inulin,such as upregulation of genes for transcription, transla-tion and protein metabolism, which could be correlatedto increased growth performance. The same studyalso reported that inulin supplementation caused theupregulation of three genes – TNFRSF1B, ACSL6 andPPARA – responsible for anti-apoptotic activity, fattyacid metabolism and energy metabolism, respectively,as well as downregulation of three genes which contrib-ute to oxidative stress, and consequently disease patho-genesis, thus showing the immunomodulatory effect ofthe prebiotic (Sevane et al. 2014). The antibacterialactivity of prebiotics is due to CE and selective promo-tion of proliferation of beneficial bacteria, which makesit difficult for pathogens to develop resistance againstthem.

Feed acidifiers

Organic acids and their salts have been used for deca-des as feed additives and are considered as GenerallyRecognized as Safe (GRAS) for meat products.Undissociated acids can diffuse through its lipid mem-brane into the bacterial cell, where they dissociate, thusdecreasing the cytoplasmic pH, consequently inhibitingnormal enzyme activity and causing cell leakage (Ricke2003). Other proposed mechanisms include disruptionof DNA (by altering purine structure), RNA and proteinsynthesis as well as interfering with cytoplasmic mem-brane structure and cell turgidity (Mani-Lopez et al.2012). Antibacterial activity of organic acids onCampylobacter spp, E. coli, Salmonella, Campylobacterperfringens and Listeria monocytogenes has beenreported (Chaveerach et al. 2002; Skrivanova et al. 2006;Over et al. 2009). Organic acids have also been studied

CRITICAL REVIEWS IN MICROBIOLOGY 325

for their effect on gut mucosa and their immunomodu-latory action. Dietary supplementation of organic acidshave shown to increase the counts of CD4 cells andT-Cell Receptor II lymphocytes, which corresponds to afaster immune response (Khan and Iqbal 2016).Additionally, organic acids are also being studied fortheir role in improvement of phytate phosphorus util-ization in chickens (Rafacz-Livingston et al. 2005). Shortchain fatty acids (SCFA) have been reported to upregu-late genes involved in epithelial cell growth, division,differentiation, proliferation and apoptosis (Hashemiand Davoodi 2011). SCFAs such as acetic acid, citricacid, lactic acid, propionic acid, butyric acid and their N,K or Ca salts are commonly included in feed as theyhave been shown to improve performance, feed qualityand modulate disease resistance of broiler (Abdel-Fattah et al. 2008; Sohail et al. 2015; Reda et al. 2016).For example, several studies have been carried out onpotential of butyric acid as feed supplement. Butyricacid has been reported to downregulate the expressionof the Salmonella pathogenicity island I genes respon-sible for virulence and invasion of epithelial cells (VanImmerseel et al. 2006). Butyrate has been shown toincrease production of tight junction proteins in thecell, thus decreasing permeability of intestinal epithe-lium to invasion by pathogens (Van Deun et al. 2008;Andreopoulou et al. 2014).

Butyrate has been studied extensively for its anti-inflammatory properties. Two pathways have been pro-posed for this: (i) inhibition of IFN-c induced STAT 1activation and (ii) inhibition of histone deacetylaseactivity leading to hyper acetylation of Fas promoter,upregulation of Fas promoter and consequently of Tcell apoptosis mediated by Fas (Zimmerman et al.2012). Another mechanism for the reduced productionof proinflammatory cytokines by butyrate is thought tobe inhibition of the activation of kappa B, a nuclear fac-tor (Onrust et al. 2015). Sunkara et al. (2011) reportedthat butyrate could induce the synthesis of hostdefence peptides in chicken, as well as increase theactivity of chicken monocytes against S. enteritidis withminimum production of inflammatory cytokines. Zhouet al. reported that sodium butyrate could modulatemacrophage activity by suppressing the production ofmatrix metalloproteinase (MMP), which causes inflam-mation. They also found that in chicken macrophage,cell lines challenged with S. typhimurium LPS, butyrateinhibited the production of IL-1b, IL-6 and IFN-c (proinflammatory cytokines) (Zhou et al. 2014)

Although organic acids are considered to be food-grade, studies have shown acid tolerance by S. typhimu-rium via the production of several acid shock proteins

and the maintenance of internal pH by amino aciddecarboxylases (Mani-Lopez et al. 2012).

Antimicrobial peptides (AMPs)

These small cationic oligopeptides are effector mole-cules of innate immunity, and have shown activityagainst bacteria, fungi and enveloped viruses (Thacker2013). It is suggested that the bactericidal activity ofAMPs is due to the formation of pores in cytoplasmicmembrane of pathogens, thus altering membrane per-meability, and, disruption of DNA and protein synthesis(Shryock and Richwine 2010). Positively charged AMPsget attracted by electrostatic interactions to structureson bacterial surfaces (e.g. lipopolysaccharide on Gram-negative walls and teichoic acids on Gram-positivewalls), and reach the cytoplasmic membrane, wherethey bind to and aggregate in the lipid bilayer, thusforming transmembrane pores leading to cell leakage,loss of osmotic regulation and cell lysis. Other possiblemechanisms of the antimicrobial activity of AMPs (afterinternalization by the cell) are activation of phospholi-pases and autolysins, inhibition of cell division by induc-ing filamentation in target cells, disruption of cell cycleand generation of reactive oxygen species (Brogden2005). AMPs have also been studied for their immuno-modulatory properties such as induction of cytokines,proliferation of cells of immune system, modulation ofgene expression, vasodilation caused by histaminerelease due to stimulation of mast cells, inhibition offibrin clot lysis to prevent pathogen dissemination, andinduction of wound and tissue repair (Finlay andHancock 2004). AMPs have also been shown to downre-gulate the lipopolysaccharide (LPS)-stimulated produc-tion of proinflammatory cytokines by suppression ofTLR signalling responses (Hancock and Sahl 2006).Studies have evaluated the antibacterial activity ofAMPs against Salmonella, Campylobacter, Listeria andEscherichia spp (Evans et al. 1995; van Dijk et al. 2007;Ebbensgaard et al. 2015), as well as the immunomodu-latory and growth promotion effects of AMPs in broilers(Yurong et al. 2006; Liu et al. 2008; Aguirre et al. 2015;J�ozefiak et al. 2016). Several AMPs produced by bacteria– called bacteriocins – have also been identified aspotential feed additives (Diez-Gonzalez 2007; Svetochet al. 2008; Xie et al. 2009; Wang et al. 2011; Messaoudiet al. 2012; Kogut et al. 2013; Perumal et al. 2016).Although there have been studies documenting devel-opment of resistance against AMPs, it is a difficult pro-cess, as AMPs target the bacterial membrane, andaltering the composition and organization of membranelipids would be not be a feasible solution for the bac-teria (Zasloff 2002).

326 G. SURESH ET AL.

Bacteriophages

One of the earliest documented uses of phage therapywas by Felix d’Herelle in 1917, when he used phagepreparations for the treatment of dysentery and chol-era. However, the extensive study on the therapeuticuse of phages gained momentum only after the emer-gence and spread of antibiotic resistance. Phage ther-apy exploits the therapeutic potential of lytic phages.These phages bind to specific receptors on bacterial cellsurface, release their genetic material into the cell anduse the host cell machinery to synthesize multiple virionparticles. Once the viruses have matured, cell wall islysed, thus releasing progeny phages (Johnson et al.2008). Several studies have reported the antibacterialpotential of bacteriophages against poultry pathogenssuch as E. coli, S. enteritidis and C. jejuni (Huff et al. 2002;Atterbury et al. 2003; Carrillo et al. 2005; Huff et al.2005; Wagenaar et al. 2005; Atterbury et al. 2007; Limet al. 2012; Hungaro et al. 2013; Seo et al. 2016). Notjust whole phages, even phage-encoded enzymes havebeen investigated for their antibacterial properties.These enzymes can be classified into virion associatedpeptidoglycan hydrolases (VAPGH) and endolysins.VAPGHs disrupt the peptidoglycan layer in bacterial cellwall after phage absorption; therefore, they are associ-ated with “lysis from without”. They produce a smallhole in the cell wall to facilitate transfer of viral DNAinto the cytoplasm. It is believed that VAPGHs have con-served, rarely modified bonds and multiple lyticdomains in their structure which development of resist-ance to these difficult (Rodr�ıguez-Rubio et al. 2013).However, limited research has been done on VAPGHsso far. Endolysins act with holins and are responsible forcell wall lysis (“lysis from within”) at the end of the lyticcycle. They hydrolyze the peptidoglycan layer in thebacterial cell wall, causing osmotic lysis and cell death,and release of the mature virions. Endolysins have acell-wall binding domain, for binding to the substrate(and cell wall debris after lysis), and enzymatically activedomains which cleave specific bonds in the peptidogly-can (Schmelcher et al. 2012). Endolysins consist ofglycosidases, amidases, carboxypeptidases and endo-peptidases (Keary et al. 2013). Genes coding for theseendolysins can be expressed in suitable vector systems.Lysins Ply 118 and Ply 511 (both amidases), whenexpressed in Lactococcos lactis, were shown to inducelysis of L. monocytogenes (Coffey et al. 2010). Anotheramidase, Ply 3626, could be a possible candidate forcontrol of C. perfringens (Fenton et al. 2010). The anti-bacterial activity of recombinant lysins against C. perfrin-gens and L. monocytogenes was also reported by Seal(2013). A chimeric, thermostable endolysin constructed

from ACP26F and AGVE2 was shown to have antibac-terial activity against C. perfringens (Swift et al. 2015).

The main drawback for the use of bacteriophages isthe development of bacterial resistance, and severalmechanisms have been proposed to explain this, suchas blocking of viral adsorption on surface receptors,degradation of viral genome by restriction-modificationsystems and phage superinfection exclusion, but thiscan be prevented by using a bacteriophage “cocktail”(Borie et al. 2014). Bacteriophages are thought to haveevolved along with the specific bacterial host; therefore,if the host develops resistance, the phages can alsoundergo mutation to overcome the resistance mechan-ism. Additionally, unlike antibiotics, the number of bac-teriophages specific for a host are not limited, henceresistance to bacteriophages is not considered as a ser-ious threat (Bragg et al. 2014).

Antibodies

Oral administration of antibodies for the development ofpassive immunity is an upcoming approach for the treat-ment of pathogens in humans as well as animals. Withrespect to poultry, egg yolk antibodies (IgY) are ofimportance as a replacement of AFAs. IgYs are maternalantibodies that the laying hens transfer to their offspringvia the egg yolk, a phenomenon that was first describedby Klemperer in 1893 (Berghman et al. 2005; Schadeet al. 2005). Antibodies specific to an antigen are pro-duced by inducing the immune system of hens by expos-ure to the antigen, and these antibodies are transferredto the egg yolk. Eventually, the egg yolk is separatedfrom the white, and antibodies are extracted from it,purified and can be utilized as a feed additive(Chalghoumi et al. 2009a; Yegani and Korver 2010).Primarily, the antibacterial activity of IgY is thought to bedue to binding of the IgY to bacterial structures such asLPS, flagella and pili, and thus preventing the adhesionto and colonization of the intestinal epithelium by thebacteria. This binding could also reduce bacterial toxinproduction by alteration of cellular signalling cascades.Other proposed mechanisms include pathogen agglutin-ation, structural changes on cell surface on binding lead-ing to increased phagocytic activity and toxinneutralization (Xu et al. 2011). Passive immunization withIgY has also been documented to increase growth andbroiler performance. Interleukin-1 (IL-1) is a cytokine thatis produced during inflammation. IL-1 further inducesproduction of Cholecystokinin (CKK), a neuropeptide thathas been found to cause anorexia growth suppression.IgYs specific to CCK have been shown to improve broilerperformance (Cook 2004; Gadde et al. 2015). Severalstudies have reported the efficacy of IgY against E. coli,

CRITICAL REVIEWS IN MICROBIOLOGY 327

Clostridium, Campylobacter and Salmonella (Tamilzarasanet al. 2009). Lee et al. (2002) and Chalghoumi et al.(2009b) demonstrated that IgY against S. enteritidis and S.typhimurium could inhibit bacterial growth in vitro.Rahimi et al. (2007) showed that chicks supplementedwith S. enteritidis-specific IgY had lower faecal sheddingand cecal colonization by the pathogen. The efficacy ofIgYs developed against C. jejuni colonization-associated-proteins (CAPs) has been demonstrated in vitro (Al-Adwani et al. 2013). Also, immunization of chickens withIgYs against whole cell lysate or hydrophobic protein ofC. jejuni has been shown to reduce C. jejuni counts in thececa after pathogen challenged. Additionally, antibodiesmay also improve feed efficacy, as reported by Mahdaviet al., that the oral administration of IgY against E. coliO78:K80 via egg yolk powder enhanced intestinal healthand broiler performance (Mahdavi et al. 2010). Comparedwith antibiotics, antigen-specific IgYs are safer, more effi-cient and less toxic. Since they are polyclonal antibodies,it is easy to develop an effective molecule without identi-fication of specific epitopes on the antigen. Althoughbacteria cannot develop resistance to antibodies, theirbiggest disadvantages are their susceptibility to proteo-lytic degradation in the gut as well as their expensivelarge-scale production (Mine and Kovacs-Nolan 2002;Gadde et al. 2015). Therefore, studies are required todevelop a simple and cost effective method of IgY pro-duction that gives high yield and purity.

Approaches for development of novelalternatives

Several properties deemed desirable for an optimal alter-native to AFA have been cited in the literature, primarily,having a well-defined mode of action, exhibiting toxicityonly to the pathogen and not the host, being non-viru-lent, not causing induction of drug resistance, cost effi-cacy, maintaining stability in feed and after industrialtreatment, improving feed efficacy and promotinggrowth, not altering palatable and being environmentallysafe (Shane 2005; Cheng et al. 2014; Caly et al. 2015).However, none of the existing alternatives individuallymeet the requisites to replace the efficiency and cost effi-cacy of AFAs. Phytogenic additives, prebiotics probiotics,synbiotics, organic acids and feed enzymes have beenreported to give inconsistent results. However, somestudies have reported that a combinational approachmay be more beneficial. Synbiotics are feed additivesthat combine both prebiotics and probiotics, whichtogether have a synergistic effect. The growth and activ-ity of the probiotic is stimulated by the prebiotic, thusbenefitting the host health and growth (Falaki et al. 2011;Mousavi et al. 2015; Saiyed et al. 2015; Tang et al. 2015;

Hamasalim 2016) However, supplementation of synbiontsdoes not always have a beneficial effect, and these incon-sistencies can be addressed by proper selection of theprobiotic strain (based on its fermentation profile) anddosage of the prebiotic (Methner et al. 1999; Geier et al.2010; Hahn-Didde and Purdum 2015; Murshed andAbudabos 2015; Abudabos et al. 2015, Flores et al. 2016).There is also documentation of recent studies of effect ofother combinations on broiler performance, such asphtyogenics and organic acids (Hassan et al. 2015;Aristimunha et al. 2016; Tanzin et al. 2016), probioticsand organic acids (Abudabos et al. 2017), and prebioticand enzymes (Rebol�e et al. 2010). A clearer understand-ing of the molecular mechanism of action of phytogenicadditives, prebiotics and probiotics would help to designmore efficient combinations of alternatives.

Another area that holds promise is the designing ofbetter delivery systems such as microencapsulation, inwhich “droplets of the bioactive compound can be sur-rounded by a coating or embedded in a homogenous orheterogeneous matrix” (Ganesh and Hettiarachchy 2015).Encapsulation is associated with increased stability, andcontrolled release of the bioactive compound in the host,and in poultry encapsulation of the bioactive compoundhas been shown reduce pathogen load, and increasecounts of beneficial bacteria (Gheisari et al. 2007; Leeet al. 2015; Rajasekaran and Santra 2015). It could be aneffective delivery system where the AFA alternative is vul-nerable to proteolytic lysis and/or low pH, such as is thecase for AMPs, phages, vaccines and antibodies.Nanoparticle delivery systems (Schlesinger et al. 2013;Adamu Ahmad et al. 2016) and gel vaccine delivery sys-tems (Jenkins et al. 2014) are more recent developmentsthat have been patented, and can be used in animalfeed. Another delivery system that is of particular interestis in ovo injection, in which the bioactive substances areinjected into the air chamber of eggs or directly into thegrowing embryo, giving the advantages of precision,control on amount of substance administered andreduced labour costs (Dankowiakowska et al. 2013). Forpoultry, in ovo injections have been documented for vac-cines against Eimeria (Barbour et al. 2015), subunit vac-cine against Campylobacter (Kobierecka et al. 2016),probiotics, prebiotic and synbiotics (Maiorano et al. 2012;Yamawaki et al. 2013; Pruszynska-Oszmalek et al. 2015;Płowiec et al. 2015), essential oil and organic acid blend(Toosi et al. 2016), etc.

Concluding remarks and future perspectives

To evade intracellular resistance mechanisms that bac-teria have developed against conventional antibiotics, itis essential to develop alternatives with different targets.

328 G. SURESH ET AL.

If the proposed alternative has multiple targets for anti-biotic activity (such as in the case of phytobiotics orAMPs), it is much harder for bacteria to develop aneffective resistance mechanism against it. Alternativessuch as probiotics and prebiotics have a host-centricmechanism of antibacterial action, i.e. they interact dir-ectly with the host gut epithelium, which in turn pre-vents colonization by pathogen. Though organic acidshave been used as feed additives for many years, resist-ance to these is not very common. This could be due tothe fact that organic acids do not bind to any specificreceptors on cell surface and their mode of entry into thecell is by simple diffusion. Similarly for AMPs, they bindto the cell surface by electrostatic attraction rather thanto a specific receptor. Bacteriophages and antibodies areemerging technologies and have their limitations; how-ever, they have high specificity for a particular host and,therefore, are superior to conventional antimicrobialswhich have an indiscriminate mode of action (i.e. againstboth pathogens and beneficial gut bacteria).

Current research suggests that for an effective AFAalternative, the focus should be on its potential immuno-modulatory efficacy, i.e. towards a host-centric approach.Management of inflammatory and immune responses inthe host facilitates the diversion of nutrients and energytowards increased production. Also, since these mecha-nisms are not directly antimicrobial, there is a lesserchance of development of resistance.

In the face of the growing population and consecu-tive increase in poultry consumption, the need of thehour is to develop a suitable alternative to AFAs. Tillsuch an alternative is developed, the judicious use ofAFAs along with improved conditions of poultry rearingis an absolute necessity.

Disclosure statement

The authors declare no conflict of interest.

Funding

The authors are sincerely thankful to the Natural Sciencesand Engineering Research Council of Canada (DiscoveryGrant 355254 and Strategic Grant), Fonds de recherche duQu�ebec – Nature et technologies (FRQNT, Projet �Equipe) forfinancial support. The views or opinions expressed in this art-icle are those of the authors.

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